Genetic alterations in the cyclin D1 gene with consequent overexpression of the encoded protein are observed in epithelial tumors of diverse histological types. Similarly, aberrant expression of the three C/EBP2 isoforms characterizes several epithelial cancers. However, the pathways through which cyclin D1 and C/EBP2 operate to effect tumorigenesis are poorly understood. A combination of bioinformatic analyses of the patterns of gene expression in human cancer and direct experimentation suggests that cyclin D1 can activate the transcriptional function of C/EBP2. Further, the transcriptional program brought about by cyclin D1 and C/EBP2 can be seen in several types of human epithelial tumors-however, the causal relationship between this transcriptional program and tumorigenesis has not been established. Research to be conducted will involve structure-function analyses to understand mechanistically how cyclin D1 activates the transcriptional function of C/EBP2 at cyclin D1 target gene promoters. The intent of these studies is also to provide reagents, e.g., C/EBP2 and cyclin D1 mutants, to probe the biological significance of the functional interaction between cyclin D1 and C/EBP2. To address the biological consequences of cyclin D1's transcriptional function, cyclin D1-dependent transformation assays have been developed, using melanocytes and mammary epithelial cells. With these systems, the contribution of cyclin D1's C/EBP2-dependent transcriptional function to transformation will be assessed genetically. A third line of investigation is inspired by the observation that cyclin D1 and C/EBP2 are required for mammary gland development during pregnancy. This observation, together with the analysis of human tumors noted above, suggests that blocked differentiation resulting from the effect of overexpressed cyclin D1 on the transcriptional activity of C/EBP2 may contribute to tumorigenesis. The research will test the possibility that the functional interaction between cyclin D1 and C/EBP2 contributes to mammary epithelial cell differentiation. This will entail a genetic analysis employing cyclin D1- and C/EBP2 -deficient mammary epithelial cells, as well as wild-type cells. PUBLIC HEALTH RELEVANCE: Cyclin D1, by virtue of its genetic alterations in human epithelial tumors-including melanoma and breast cancer-is thought to contribute materially to cancer. In order to target cyclin D1 therapeutically, it is necessary to understand the pathways through which it operates. Responding to this need, the proposed research will attempt to elucidate how cyclin D1 effects tumorigenesis.